ABSTRACT
Emerald ash borer (Agrilus planipennis; EAB) has devastated populations of ash (Fraxinus spp.) trees in dozens of U.S. states and Canada over the past few decades. The continued survival of scattered ash trees known as "lingering ash" in heavily infested natural stands, however, offers evidence of genetic resistance or tolerance to EAB. These surviving or "lingering" ash individuals may form the basis for reforestation programs in EAB-impacted areas, and clonal mass-propagation of these genotypes can help accelerate these efforts. Between 2013 and 2018, we initiated embryogenic cultures by culturing immature zygotic embryos from open-pollinated (OP) seeds collected from several surviving white ash and green ash trees in Michigan and Pennsylvania. In addition, in 2018, we initiated cultures from crosses made between lingering green ash parents from the USDA Forest Service ash breeding program in Ohio. Somatic embryos were produced by growing cultures in liquid suspension, followed by fractionation and plating on semisolid medium to produce developmentally synchronous populations of somatic embryos. Somatic embryo germination and conversion were enhanced by a combination of pre-germination cold treatment and inclusion of activated charcoal and gibberellic acid in the germination medium. Ash somatic seedlings derived from OP explants grew rapidly following transfer to potting mix and somatic seedlings representing nine ash clones were acclimatized, grown in the greenhouse and planted in a preliminary field test, along with EAB-resistant Manchurian ash (F. mandshurica) and EAB-susceptible control seedlings. Somatic seedlings have now been produced from cultures that originated from seeds derived from the progeny of lingering green ash parents and an ex vitro germination protocol has shown some promise for accelerating early somatic seedling growth. Results of this research could provide the basis for scaled-up production of EAB-resistant ash varieties for seed orchard production for forest restoration and cultivar development for urban tree restoration.
ABSTRACT
Embryogenic cultures of loblolly pine (Pinus taeda L.), slash pine (Pinus elliottii Engelm.), longleaf pine (Pinus palustris Mill.) and slash pine x longleaf pine hybrids were initiated from immature seeds on an initiation medium containing 13.57 microM 2,4-dichlorophenoxyacetic acid and 2.22 microM benzylaminopurine. Embryogenic cultures proliferated and somatic embryos developed, matured and germinated following a modified protocol and media originally developed for radiata pine (Pinus radiata D. Don.) somatic seedling production. A discrete, light-sensitive pre-germination stage and a later germination (radicle emergence) stage were identified by the differential response of somatic embryos to light of different wavelengths. Different light quality treatments were applied during the pre-germination and germination steps, using cool white fluorescent bulbs or light-emitting diodes (LEDs), or both. In general, red wavelengths provided by LEDs during these steps resulted in higher frequencies of somatic embryo germination (up to 64%) and conversion (up to 50%), longer tap roots and more first-order lateral roots than the standard cool white fluorescent treatments or treatment with blue wavelengths from LEDs. In addition, exposure to red light allowed germination of somatic embryos of some clones that failed to produce germinants under fluorescent light. Germination and conversion were further enhanced by sequential application of cool white fluorescent light and red light, resulting in up to 100% germination and conversion in one experiment. Longleaf pine somatic embryos were especially responsive to the light quality treatments, resulting in the first report of somatic seedling production for this species.